Technical updates to basic proteins focalization using IPG strips

Abstract

Background: Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility.

Results: Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6-11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks.

Conclusion: According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.

Figure 1

2DE separation of MCF7 basic protein: iso-electric focalisation was performed with 50 μg proteins using 6–11 pH IPG strip and 12% acrylamide was used for the second dimension. Proteins were loaded by in-gel rehydration (A), anodic cup loading (B), and paper-bridge loading (C).

Figure 2

Comparison of MCF7 basic protein 2DE separation using twelve different anodic cup-loading protocols. Iso-electric focalisation was performed with 50 μg proteins using 6–11 pH IPG strip and 12% acrylamide was used for the second dimension. The number reported on each picture corresponded to the cup-loading condition used, as presented in detail in Table 1.

Figure 3

2DE map of MCF7 basic protein separation using anodic cup-loading protocol n°9, as presented in Figure 2 and Table 1. Specific areas A and B were further analysed.

Figure 4

Picture comparison of areas A and B from Figure 3 as a function of different anodic cup-loading protocols. The number of spots within each area was estimated by picture analysis and reported in Table 1.

Figure 5

2DE map comparison and alignment of bovine milk proteins using in-gel rehydration or paper-bridge loading. 600 μg of non-fat dry milk proteins were in-gel loaded using 3–10 IPG strips (A) or paper-bridge loaded using 6–11 IPG strips with (C) or without electrode wicks (D) during focalization. In a second time, paper-bridge previously used with 6–11 IPG strip was applied with 4–7 IPG strips (B) to focalize remaining proteins.

Figure 6

Schematic of IPG strip, electrodes and paper wicks positions as function of protein loading protocols. A: sample in-gel rehydration of IPG strip, with acrylamide face down. B: cup-loading of protein sample, with acrylamide face up, a plastic sample cup was placed at the anodic end, paper wicks connected strip with electrodes.

Figure 7

Schematic of IPG strip placement before the second dimension. A special precaution is presented to seal strips with 1% agarose and curved plastic pieces.